P53 Wild-type Colon Cancer Research Articles

Glyceollins Modulate Tumor Development and Growth in a Mouse Xenograft Model of Human Colon Cancer in a p53-Dependent Manner.

Glyceollins are soybean-derived phytoalexins that induce the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway, which is involved in the detoxification of carcinogens and the removal of reactive oxygen species (ROS). Recent studies, however, have indicated that Nrf2 induction stimulates the development of pre-existing tumors and confers resistance to chemotherapy by elevating drug metabolism and by efficient scavenging of ROS produced by the Warburg effect, which is regulated, in turn, by the p53 tumor suppressor. This study, therefore, aimed at examining whether glyceollins could accelerate tumor growth in the presence of active p53, using a xenograft BALB/c nude mouse model transplanted subcutaneously with p53 wild-type and p53 null HCT116 human colon cancer cells. Glyceollins were orally administered at a dose of either 1 or 4 mg/kg body weight after xenografting HCT116 cells, and tumor growth and volume were monitored for 2 weeks. A high dose of glyceollins resulted in a significant increase in the average volume of p53 wild-type HCT116 xenografts, but not of p53 null HCT116 xenografts. However, a low dose of glyceollins had no effect on the tumor growth regardless of p53 presence. Interestingly, antioxidant enzymes, including heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase, were prominently induced by glyceollins in p53 wild-type xenografts, compared with p53 null xenografts. These results suggest that a high dose of glyceollins possibly promotes the growth of p53 wild-type colon cancer through activation of the Nrf2-mediated signaling pathway and, in particular, strong induction of HO-1 expression. Therefore, the consumption of Nrf2 activators, including glyceollins, should be carefully monitored for patients suffering from certain types of cancer and/or receiving chemotherapy.

Kruppel-like factor 2 mediated anti-proliferative and anti-metastasis effects of simvastatin in p53 mutant colon cancer

The changes in cellular metabolism play an important role in promoting tumor progression. Recent findings suggested that the mutation of tumor suppressor gene p53 promoted lipids synthesis and mutant p53 (mutp53) was essential for regulating mevalonate pathway for cholesterol synthesis. Simvastatin, a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, was found to exhibit therapeutic effects against many types of cancers including breast cancer, colon cancer, lung cancer, etc. However, the underlying mechanism of the antitumor effect of simvastatin still needs to be further investigated. Our data demonstrated that suppression of mevalonate pathway by simvastatin significantly upregulated Kruppel-like factor 2 (KLF2) and p21WAF1/CIP1 expression in mutp53 colon cancer cells SW1116 but not in p53 wild type cells HCT116. Meanwhile, we found that overexpression of KLF2 could significantly induce p21WAF1/CIP1 expression, inhibit Wnt signaling and suppress epithelial-mesenchymal transition, indicating that KL2 might mediate antitumor effect of simvastatin in SW1116 cells. Moreover, bioinformatic analysis from The Cancer Genome Atlas (TCGA) database indicated that KLF2 were positively correlated with CDKN1A (encoding p21WAF1/CIP1), both of which were downregulated in colon cancer tissue, especially in p53 mutant colon cancer tissue. The results showed that KLF2 might be a tumor suppressor gene in colon cancer, which was in accordance with our experimental data. We also found that CDKN1A expression in mutant p53 colon cancer tissue was significant decreased when compared with p53 wild type colon cancer tissue, while Wnt ligand Wnt5a exhibited the highest level in p53 mutant colon cancer tissue. These data provide strong evidences for clinical application of simvastatin in treatment of colon cancer with p53 mutation.

Leptadenia pyrotechnica Induces P53-Dependent Apoptosis in Colon Cancer Cells

Leptadenia pyrotechnica has been commonly used traditionally for the treatment of tumors in Gulf countries. However, the molecular mechanism of the anti-cancer activity of Leptadenia pyrotechnica remains unclear. In the present study, 80% ethanolic extract of Leptadenia pyrotechnica and its fractions (n-hexane, EA, n-butanol and aqueous) were evaluated for in vitro cytotoxicity against colon cancer cell lines (HCT 116 wild type and HCT 116 p53−/−). The hexane fraction of Leptadenia pyrotechnica (LHE) is identified as the major anti-cancer fraction in Leptadenia pyrotechnica solvent extracts. Cells were incubated with different concentrations of LHE. Cell viability was quantified by crystal violet and MTT assays. Apoptotic cells were determined using Annexin V-propidium iodide (Annexin V/PI) staining and γH2AX assay for damaged DNA. Bax and Bcl-2, caspase-8, 9, 3 protein expression was analyzed by western blotting. LHE decreased cell viability in colon cancer cells in a dose- and time-dependent manner with an IC50 of 100 μg/ml after 24 h. Further experiments on colon cancer cells treated with LHE at 100 μg/ml for 24 h and 48 h revealed that it induces a p53-dependent apoptosis through intrinsic as well as extrinsic pathways. Analysis of DNA fragmentation by flow cytometry and H2AX assay showed a higher apoptotic cell death in p53 wildtype colon cancer cells treated with LHE in comparison to p53-/- cells. Data of western blots further demonstrated that LHE treatment caused activation of caspases, up regulation of Bax and down regulation of Bcl-2 in a time-dependent manner in HCT 116 cell lines. GC-MS analysis of LHE revealed the presence of derivatives of linoleic acid (octadecadienoic), diterpenes (phytol), triterpenes (squalene), and lupeol as the major constituents. These findings collectively suggest that LHE induces apoptosis in HCT 116 cells possibly mediated by ROS generation and involved multiple pathways including extrinsic, intrinsic and caspase-mediated pathways. Our results suggest that LHE may be applied as a potential anti-cancer drug against human colon cancer having intact p53 function.

MiR-34a-5p suppresses colorectal cancer metastasis and predicts recurrence in patients with stage II/III colorectal cancer.

Although surgery remains the mainstay of curative treatment for colorectal cancer (CRC), many patients still have high chance to experience disease relapse. It is therefore imperative to identify prognostic markers that can help predict the clinical outcomes of CRC. Aberrant microRNA expression holds great potential as diagnostic and prognostic biomarker for CRC. Here we aimed to investigate clinical potential of miR-34a-5p as a prognostic marker for CRC recurrence and its functional significance. First, we validated that miR-34a-5p was downregulated in CRC tumour tissues (P<0.05). The expression level of tissue miR-34a-5p was then evaluated in two independent cohorts of 268 CRC patients. miR-34a-5p expression was positively correlated with disease-free survival in two independent cohorts (cohort I: n=205, P<0.001; cohort II: n=63, P=0.006). Moreover, the expression of miR-34a-5p was an independent prognostic factor for CRC recurrence by multivariate analysis (P<0.001 for cohort I, P=0.007 for cohort II). Ectopic expression of miR-34a-5p in p53 wild-type colon cancer cell HCT116 significantly inhibited cell growth, migration, invasion and metastasis. miR-34a-5p induced cell apoptosis, cell cycle arrest at G1 phase and p53 transcription activity in HCT116 cells, but not in the HCT116 p53 knockout (p53(-/-)) cells. miR-34a-5p significantly suppressed the HCT116 growth in vivo, whereas it showed no effect on the HCT116 p53(-/-) xenograft, indicating that the growth-inhibiting effect by miR-34a-5p was dependent on p53. In addition, the expression level of miR-34a-5p in patients with p53-positive expression was higher than that in patients with p53-negative expression (P<0.01). In conclusion, miR-34a-5p inhibits recurrence of CRC through inhibiting cell growth, migration and invasion, inducing cell apoptosis and cell cycle arrest in a p53-dependent manner.

Overexpression of miR-22 reverses paclitaxel-induced chemoresistance through activation of PTEN signaling in p53-mutated colon cancer cells

Chemoresistance is a key cause of treatment failure in colon cancer. MiR-22 is a tumor-suppressing microRNA. To explore whether miR-22 is an important player in the development of chemoresistance in colon cancer, we overexpressed miR-22 and subsequently tested its role in cell proliferation, apoptosis, survival, and associated signaling in p53-mutated HT-29 and HCT-15 cells, and p53 wild-type HCT-116 cells. We further investigated the role of miR-22 on cytotoxicity of paclitaxel in both the p53-mutated and p53 wild-type colon cancer cells. Results showed that HT-29 and HCT-15 cells were resistant to paclitaxel-induced cytotoxicity, which normally inhibits cell proliferation and survival, and induces apoptosis. Conversely, HCT-116 was relatively sensitive to the cytotoxicity of paclitaxel. Overexpression of miR-22 significantly decreased cell proliferation and survival, and induced cell apoptosis in the p53-mutated colon cancer cells, but played no role in the p53 wild-type cells. Importantly, miR-22 overexpression enhanced the cytotoxic role of paclitaxel in p53-mutated HT-29 and HCT-15 cells, but not in p53 wild-type HCT-116 cell. We further demonstrated that the tumor-suppressive role of miR-22 in p53-mutated colon cancer cells was mediated by upregulating PTEN expression, which negatively regulated Akt phosphorylation at Ser(473) and MTDH expression, and subsequently increased Bax and active caspase-3 levels. Our study is the first to identify the tumor-suppressive role of miR-22 and its associated signaling in the p53-mutated colon cancer cells and highlighted the chemosensitive role of miR-22.

Abstract 5486: Combined targeting of the PI3K and MAPK pathways is required for efficacy in treating colorectal cancer of the wild and mutant types

Abstract The KRAS oncogene is commonly mutated in human colon cancer. The downstream pathways of mutant KRAS, including the RAF/MEK/ERK, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)-AKT signaling are indispensible in order to understand how to pharmacologically target them when treating colon cancer with the KRAS mutant. However, the heterogeneous responses of these effector pathways remain unclear. Recent studies have indicated that mutational activation of PI3K (PIK3CA) in KRAS mutant tumor cells induces resistance to the inhibition of MEK, one of the MAPK signal cascades. Wild-type p53 also suppresses the oncogenic potential of the MAPK pathway. Our study shows that bortezomib inhibits the PI3K-AKT pathway and that nutlin-3A used as a p53-inducing agent blocks the MAPK pathway. The high doses of bortezomib induced apoptosis through complete suppression of AKT phosphorylation in KRAS and PI3K mutant colon cancer cells. Treating patients using high doses of nutlin-3A resulted in apoptosis by inducing p53 phosphorylation in colon cancer cells with wild-type p53. We also showed that the combination of bortezomib and nutlin-3A exhibited a synergistic effect on KRAS mutant colon cancer cells with PI3K mutant and wild-type p53, whereas there are no combined effect in KRAS mutant colon cancer cells with the p53 mutant. In addition, bortezomib and nutlin-3A exhibited combined effect in KRAS wild-type colon cancer cells with PI3K mutant and wild-type p53, although not in p53 mutant cells. We consistently noted that treatment with bortezomib and nutlin-3A after p53 silencing using small, interfering RNA (siRNA) in p53 wild-type colon cancer cells, resulted in a decrease of the populations of dead cells. Therefore, these results suggest that a combination of bortezomib and nutlin-3A can overcome the heterogeneous responses of the effector pathway in colon cancer with KRAS mutant; they also provide a new basis for the clinical testing of PI3K- and MAPK-targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2011-5486

IGF‐1R Regulation of Colon Cancer Stem Cells

Despite recent advances in therapeutics, recurrence of colon cancer occurs in 50% of the cases, which could partly be due to the presence of self renewing cancer stem‐like cells (CSCs) which are resistant to chemotherapy. We have observed that chemosurviving colon cancer cells are enriched in CSCs as evidenced by increased number of CD44+ cells, associated with activated growth factor receptors especially IGF‐1R (4.5‐fold). Depletion of IGF‐1R by si‐RNA results in growth inhibition of chemosurviving cells accompanied by a reduction in CD44 suggesting that IGF‐1R might target CSCs. Indeed, we observed a significant inhibition of colonosphere formation, a CSC property, in IGF‐1R si‐RNA transfected cells in p53 wt HCT‐116 cells accompanied by decrease in CD44 and CD166 expression and induction in miRNA which post transcriptional regulate gene expression. We identified miRNA‐363, a non‐p53 regulated miRNA, to be highly overexpressed (18.9‐fold) following IGF‐1R inhibition. In addition, miR‐215, a p53 regulated miRNA, which is normally downregulated in colon cancer, was upregulated by 2‐fold following IGF‐1R inhibition resulting in induction of its downstream effector p21waf1/cip1 only in p53 wild type colon cancer cells. We conclude that Inhibition of IGF‐1R results in reduction of CSC phenotype in colorectal cancer cells which could partly be due to induction of miRNAs ‐363 and ‐215.

P53-Mediated upregulation of DcR1 impairs oxaliplatin/TRAIL-induced synergistic anti-tumour potential in colon cancer cells

Oxaliplatin has emerged as a major chemotherapeutic drug in the treatment of advanced colorectal cancer, yet like most conventional cancer therapeutics, its efficacy is often compromised due to p53 mutations. Unlike oxaliplatin, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a p53-independent manner, and chemotherapy is known to overcome tumour resistance to TRAIL-induced cell death in most cancer cells. Using a panel of colon cancer cell lines, we assessed the ability of oxaliplatin to sensitize to TRAIL-induced apoptosis. We demonstrate that while both drugs additively or synergistically induced apoptosis in almost all cell lines tested, p53 wild-type colon cancer cells such as HCT116, LS513 or LS174T remained resistant. Impaired TRAIL-induced cell death resulted from a strong p53 dependent, oxaliplatin-mediated, DcR1 receptor expression increase. According to our finding, downregulation of DcR1 using siRNA, in p53 wild-type colon cancer cells, restored oxaliplatin/TRAIL synergistic apoptotic activity. On the contrary, exogenous DcR1 overexpression in SW480, a p53-mutated cell line, abolished the synergy between the two drugs. Altogether we demonstrate for the first time that p53 negatively regulates oxaliplatin-mediated TRAIL-induced apoptotic activity through DcR1 upregulation. Our findings could have important implications for future therapeutic strategies, and suggest that the association oxaliplatin/TRAIL should be restricted to patients harbouring a non-functional p53 protein.

P53 dependent and independent sensitivity to oxaliplatin of colon cancer cells

Oxaliplatin is an efficient chemotherapeutic agent used for the treatment of metastatic human colon cancer, but cancer cells are frequently resistant. The aim of this study was to analyse the underlying mechanisms in a panel of 10 human colorectal cancer cell lines submitted to a short (2h) oxaliplatin treatment period, accordingly to the usual therapeutic procedure in humans. Sensitivity to oxaliplatin was a characteristic of p53 wild-type colon cancer cells. In contrast, all p53-mutated cell lines had a high IC50 to oxaliplatin, with the exception of the V9P cell line. Exposure to oxaliplatin resulted in G0/G1 arrest in p53 wild-type cell lines, and in S phase in p53-mutated cell lines. In our treatment conditions, no DNA accumulation in sub G0/G1 phase, no caspase-3 activation nor PARP cleavage were detected after oxaliplatin treatment, except for the V9P cell line. The major role of the p53–p21 pathway in oxaliplatin sensitivity was confirmed in the p53 wild-type HCT116 cell line, using siRNA duplex, and knockdown of the TAp73 protein also enhanced resistance to oxaliplatin in this cell line. Surprisingly, siRNA duplex invalidation revealed a residual effect of the mutant p53 protein in p53-mutated cell lines. Persistent sensitivity to oxaliplatin of the p53-mutated V9P cell line was associated with oxalipatin-induced apoptosis but TAp73 was not the responsible alternative pathway.